The quantification of chemical and biochemical components in colored aqueous fluids, in particular colored biological fluids such as whole blood and urine and biological fluid derivatives such as serum and plasma, is of ever-increasing importance. Important applications exist in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals and the like. In some instances, the amounts of materials being determined are either so miniscule--in the range of a microgram or less per deciliter--or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case, the results are generally not available for some hours or days after sampling. In other instances, there is often an emphasis on the ability of lay operators to perform the test routinely, quickly and reproducibly outside a laboratory setting with rapid or immediate information display.
One common medical test is the measurement of blood glucose levels by diabetics. Current teaching counsels diabetic patients to measure their blood glucose level from two to seven times a day depending on the nature and severity of their individual cases. Based on the observed pattern in the measured glucose levels the patient and physician together make adjustments in diet, exercise and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.
Previously, a method widely used in the United States employs a test article of the type described in U.S. Pat. No. 3,298,789 issued Jan. 17, 1967 to Mast. In this method a sample of fresh, whole blood (typically 20-40 .mu.l) is placed on an ethylcellulose-coated reagent pad containing an enzyme system having glucose oxidase and peroxidase activity. The enzyme system reacts with glucose and releases hydrogen peroxide. The pad also contains an indicator which reacts with the hydrogen peroxide in the presence of peroxidase to give a color proportional in intensity to the sample's glucose level.
Another previous blood glucose test method employs similar chemistry but in place of the ethylcellulose-coated pad employs a water-resistant film through which the enzymes and indicator are dispersed. This type of system is disclosed in U.S. Pat. No. 3,630,957 issued Dec. 28, 1971 to Rey et al.
In both cases the sample is allowed to remain in contact with the reagent pad for a specified time (typically one minute). Then in the first case the blood sample is washed off with a stream of water while in the second case it is wiped off the film. The reagent pad or film is then blotted dry and evaluated. The evaluation is made either by comparing color generated with a color chart or by placing the pad or film in a diffuse reflectance instrument to read a color intensity value.
While the above methods have been used in glucose monitoring for years, they do have certain limitations. The sample size required is rather large for a finger stick test and is difficult to achieve for some people whose capillary blood does not express readily.
In addition, these methods share a limitation with other simple lay-operator colorimetric determinations in that their result is based on an absolute color reading which is in turn related to the absolute extent of reaction between the sample and the test reagents. The fact that the sample must be washed or wiped off the reagent pad after the timed reaction interval requires that the user be ready at the end of the timed interval and wipe or apply a wash stream at the required time. The fact that the reaction is stopped by removing the sample leads to some uncertainty in the result, especially in the hands of the home user. Overwashing can give low results and underwashing can give high results.
Another problem that often exists in simple lay-operator colorimetric determinations is the necessity for initiating a timing sequence when blood is applied to a reagent pad. A user will typically have conducted a finger stick to a obtain a blood sample and will than be required to simultaneously apply the blood from the finger to a reagent pad while initiating a timing circuit with his or her other hand, thereby requiring the use of both hand simultaneously. This is particularly difficult since it is often necessary to insure that the timing circuit is started only when blood is applied to the reagent pad. In order to eliminate the need for the user to initiate a timing sequence upon application of the blood sample to the reagent pad, U.S. Pat. No. 5,049,487 issued Sep. 17, 1991 to Phillips et al. teaches the use of a reagent pad and reflectance measurement system as illustrated schematically in FIG. 1. The Phillips et al. patent teaches an apparatus for determining the presence of an analyte in a fluid as well as a test strip for use with the apparatus. The fluid to be analyzed is applied to the test strip and the test strip is analyzed by the apparatus. In a preferred embodiment, the test strip comprises a single layer hydrophilic porous matrix 10 to which the chemical reagents are bound. The chemical reagents react with the analyte in the sample applied to the test strip in order to produce a dye that is characteristically absorptive at a wavelength other than the wavelength that the assay medium substantially absorbs. In other works, reaction of the chemical reagent with the analyte produces a color change in the sample.
The reagent matrix 10 is coupled to the underside of an inert test strip carrier 12 containing an orifice 14 therethrough. The analyte sample is applied to the orifice 14 and the apparatus analyzes the opposite side of the test strip by reflecting light from an LED 16 off of the bottom surface of the reagent matrix 10 and sensing the amount of reflected light with a photodiode 18. It is therefore necessary for the sample to diffuse through the test strip prior to being analyzed. In such systems, the amount of time that the analyte is allowed to react with the reagent prior to measurement of a color change is critical to the accuracy of the measurement. The beginning of this "incubation period" must be measured as precisely as possible. In the Phillips et al. patent, as the analyte sample penetrates the reagent matrix 10 and wets the bottom surface, an initial change in reflectance of this measurement surface occurs. The apparatus detects this change in reflectance by sensing a decrease in the amount of light reflected to the photodetector 18. The apparatus then begins the incubation period upon detection of this change in reflectance. After a predetermined incubation time period, during which the sample containing the analyte reacts with the reagent chemicals in the matrix 10, a second reflectance measurement is made in order to determine the color change in the sample. By accurately measuring the beginning of the incubation period and the time delay before measurement, the accuracy of the apparatus is greatly improved over prior methods.
As shown in FIG. 2, the Phillips et al. method could also be adapted to an electrochemical measurement system, in which changes in conductivity of the reagent pad 10 are measured by a current meter 20. Two electrodes 22, 24 are placed in contact with the reagent pad 10 and a voltage source 26 is coupled across the electrodes 22, 24. The amount of current measured by the current meter 20 is directly proportional to the conductivity of the reagent pad 10, which conductivity changes as the blood glucose reacts with the reagent.
Because the start of the incubation period in the Phillips et al. method begins with a determination that surface wetting of the underside of the reagent matrix 10 has occurred (in the embodiment of FIG. 1), the processing circuitry coupled to the photodetector 18 must have some method for determining when the reflectance measurements indicate surface wetting. Referring to FIG. 3, there is shown a graph of remission (percent reflection) v. the apparatus system time (in which one unit of system time equals 0.25 seconds of actual time). As can be seen from the graph, the reflectivity of the reagent matrix 10 prior to sample application is a constant value (approximately 88%). After sample application, the reflectivity of the underside of the reagent matrix 10 steadily drops as the sample fluid migrates to the underside of the reagent matrix 10. The remission also drops due to a color change in the reagent caused by reaction with the analyte sample. At some point, the analyte fluid has reached the undersurface of the reagent matrix 10 and further drops in remission are caused only by color change of the reagent. The prior art method analyzes this data in order to make a determination of when surface wetting has occurred on the underside of the reagent matrix 10. This determination is made by sensing when the remission value has dropped by a predetermined, fixed amount from its steady state value prior to sample application. For example, in one commercial version of this prior art system, surface wetting is assumed to have occurred when the remission value drops by approximately 38% (i.e. when a remission value of 50% is observed). When this change in remission (or .DELTA.R) is observed, the prior art device starts the timing of the incubation period, after which the sample measurement will be made.
As shown in FIG. 4, the prior art Phillips et al. method can be adapted to the electrochemical sensor apparatus of FIG. 2, since the measured current exhibits a sharp increase upon application of the sample. The start of the incubation period may therefore be started when the current increases by more than a predetermined, fixed amount.
The prior art method described in Phillips et al. suffers from the problem that the start of the incubation period, by using a fixed reflectance drop, must be tailored to a specific chemistry. A changing in the base reflectance, such as may occur for different enzymes or indicators, requires a predetermination of the fixed reflectance drop. In this sense, a given fixed drop is not generally applicable to different systems.